The Challenges and Emerging Opportunities of Targeting Cytokines and Chemokine-Driven Inflammatory Signals in Metastatic Castrate-Resistant Prostate Cancer.

Inflammation is a key risk factor and functional driver in the initiation and progression of prostate cancer (PCa). De-regulated cytokine and chemokine signaling facilitates critical communication between tumor cells and multiple cell lineages within the tumor microenvironment (TME). Historical attempts at using targeted approaches to disrupt inflammation have been disappointing, with sub-optimal or negligible clinical benefit. Our increased awareness of the myeloid infiltrate in supporting the acquisition of castrate resistance and underpinning the abject response of advanced PCa to immunotherapy has re-focused attention on improved strategies to disrupt these complex cytokine and chemokine signaling networks within the TME. These ongoing and prospective strategies are principally focused on employing cytokine-/chemokine-directed therapies in informed combination with androgen signaling inhibitors or immunotherapeutic agents and, increasingly, with due consideration of the genetic context of the tumor. The availability of molecular-targeted therapeutic agents directed against the critical signal transduction nodes activated by cytokine and chemokine signaling in tumor cells provides opportunities to reduce the impacts of biological redundancy. Precision-based trials that deploy this latest generation of cytokine- and chemokine-directed therapeutics, directed to enriched patient cohorts in a biologically informed and biomarker-guided manner, have the potential to diversify the armamentarium of agents that is required in order to transform long-term outcomes for a currently incurable and genetically heterogenous disease.

Combined impact of lipidomic and genetic aberrations on clinical outcomes in metastatic castration-resistant prostate cancer.

BACKGROUND: Both changes in circulating lipids represented by a validated poor prognostic 3-lipid signature (3LS) and somatic tumour genetic aberrations are individually associated with worse clinical outcomes in men with metastatic castration-resistant prostate cancer (mCRPC). A key question is how the lipid environment and the cancer genome are interrelated in order to exploit this therapeutically. We assessed the association between the poor prognostic 3-lipid signature (3LS), somatic genetic aberrations and clinical outcomes in mCRPC. METHODS: We performed plasma lipidomic analysis and cell-free DNA (cfDNA) sequencing on 106 men with mCRPC commencing docetaxel, cabazitaxel, abiraterone or enzalutamide (discovery cohort) and 94 men with mCRPC commencing docetaxel (validation cohort). Differences in lipid levels between men ± somatic genetic aberrations were assessed with t-tests. Associations between the 3LS and genetic aberrations with overall survival (OS) were examined using Kaplan-Meier methods and Cox proportional hazard models. RESULTS: The 3LS was associated with shorter OS in the discovery (hazard ratio [HR] 2.15, 95% confidence interval [CI] 1.4-3.3, p < 0.001) and validation cohorts (HR 2.32, 95% CI 1.59-3.38, p < 0.001). Elevated plasma sphingolipids were associated with AR, TP53, RB1 and PI3K aberrations (p < 0.05). Men with both the 3LS and aberrations in AR, TP53, RB1 or PI3K had shorter OS than men with neither in both cohorts (p ≤ 0.001). The presence of 3LS and/or genetic aberration was independently associated with shorter OS for men with AR, TP53, RB1 and PI3K aberrations (p < 0.02). Furthermore, aggressive-variant prostate cancer (AVPC), defined as 2 or more aberrations in TP53, RB1 and/or PTEN, was associated with elevated sphingolipids. The combination of AVPC and 3LS predicted for a median survival of ~12 months. The relatively small sample size of the cohorts limits clinical applicability and warrants future studies. CONCLUSIONS: Elevated circulating sphingolipids were associated with AR, TP53, RB1, PI3K and AVPC aberrations in mCRPC, and the combination of lipid and genetic abnormalities conferred a worse prognosis. These findings suggest that certain genotypes in mCRPC may benefit from metabolic therapies.

Attenuating Adaptive VEGF-A and IL8 Signaling Restores Durable Tumor Control in AR Antagonist-Treated Prostate Cancers.

Inhibiting androgen signaling using androgen signaling inhibitors (ASI) remains the primary treatment for castrate-resistant prostate cancer. Acquired resistance to androgen receptor (AR)-targeted therapy represents a major impediment to durable clinical response. Understanding resistance mechanisms, including the role of AR expressed in other cell types within the tumor microenvironment, will extend the clinical benefit of AR-targeted therapy. Here, we show the ASI enzalutamide induces vascular catastrophe and promotes hypoxia and microenvironment adaptation. We characterize treatment-induced hypoxia, and subsequent induction of angiogenesis, as novel mechanisms of relapse to enzalutamide, highlighting the importance of two hypoxia-regulated cytokines in underpinning relapse. We confirmed AR expression in CD34+ vascular endothelium of biopsy tissue and human vascular endothelial cells (HVEC). Enzalutamide attenuated angiogenic tubule formation and induced cytotoxicity in HVECs in vitro, and rapidly induced sustained hypoxia in LNCaP xenografts. Subsequent reoxygenation, following prolonged enzalutamide treatment, was associated with increased tumor vessel density and accelerated tumor growth. Hypoxia increased AR expression and transcriptional activity in prostate cells in vitro. Coinhibition of IL8 and VEGF-A restored tumor response in the presence of enzalutamide, confirming the functional importance of their elevated expression in enzalutamide-resistant models. Moreover, coinhibition of IL8 and VEGF-A resulted in a durable, effective resolution of enzalutamide-sensitive prostate tumors. We conclude that concurrent inhibition of two hypoxia-induced factors, IL8 and VEGF-A, prolongs tumor sensitivity to enzalutamide in preclinical models and may delay the onset of enzalutamide resistance. IMPLICATIONS: Targeting hypoxia-induced signaling may extend the therapeutic benefit of enzalutamide, providing an improved treatment strategy for patients with resistant disease.

Modulating the unfolded protein response with ONC201 to impact on radiation response in prostate cancer cells.

Prostate cancer (PCa) is the most common non-cutaneous cancer in men and a notable cause of cancer mortality when it metastasises. The unfolded protein response (UPR) can be cytoprotective but when acutely activated can lead to cell death. In this study, we sought to enhance the acute activation of the UPR using radiation and ONC201, an UPR activator. Treating PCa cells with ONC201 quickly increased the expression of all the key regulators of the UPR and reduced the oxidative phosphorylation, with cell death occurring 72 h later. We exploited this time lag to sensitize prostate cancer cells to radiation through short-term treatment with ONC201. To understand how priming occurred, we performed RNA-Seq analysis and found that ONC201 suppressed the expression of cell cycle and DNA repair factors. In conclusion, we have shown that ONC201 can prime enhanced radiation response.

Prostate cancer heterogeneity assessment with multi-regional sampling and alignment-free methods.

Combining alignment-free methods for phylogenetic analysis with multi-regional sampling using next-generation sequencing can provide an assessment of intra-patient tumour heterogeneity. From multi-regional sampling divergent branching, we validated two different lesions within a patient's prostate. Where multi-regional sampling has not been used, a single sample from one of these areas could misguide as to which drugs or therapies would best benefit this patient, due to the fact these tumours appear to be genetically different. This application has the power to render, in a fraction of the time used by other approaches, intra-patient heterogeneity and decipher aberrant biomarkers. Another alignment-free method for calling single-nucleotide variants from raw next-generation sequencing samples has determined possible variants and genomic locations that may be able to characterize the differences between the two main branching patterns. Alignment-free approaches have been applied to relevant clinical multi-regional samples and may be considered as a valuable option for comparing and determining heterogeneity to help deliver personalized medicine through more robust efforts in identifying targetable pathways and therapeutic strategies. Our study highlights the application these tools could have on patient-aligned treatment indications.

Clinical and functional characterization of CXCR1/CXCR2 biology in the relapse and radiotherapy resistance of primary PTEN-deficient prostate carcinoma.

Functional impairment of the tumour suppressor PTEN is common in primary prostate cancer and has been linked to relapse post-radiotherapy (post-RT). Pre-clinical modelling supports elevated CXC chemokine signalling as a critical mediator of PTEN-depleted disease progression and therapeutic resistance. We assessed the correlation of PTEN deficiency with CXC chemokine signalling and its association with clinical outcomes. Gene expression analysis characterized a PTEN LOW/CXCR1HIGH/CXCR2HIGH cluster of tumours that associates with earlier time to biochemical recurrence [hazard ratio (HR) 5.87 and 2.65, respectively] and development of systemic metastasis (HR 3.51). In vitro, CXCL signalling was further amplified following exposure of PTEN-deficient prostate cancer cell lines to ionizing radiation (IR). Inhibition of CXCR1/2 signalling in PTEN-depleted cell-based models increased IR sensitivity. In vivo, administration of a CXCR1/2-targeted pepducin (x1/2pal-i3), or CXCR2-specific antagonist (AZD5069), in combination with IR to PTEN-deficient xenografts attenuated tumour growth and progression compared to control or IR alone. Post-mortem analysis confirmed that x1/2pal-i3 administration attenuated IR-induced CXCL signalling and anti-apoptotic protein expression. Interventions targeting CXC chemokine signalling may provide an effective strategy to combine with RT in locally advanced prostate cancer patients with known presence of PTEN-deficient foci.

ATM Kinase Inhibition Preferentially Sensitises PTEN-Deficient Prostate Tumour Cells to Ionising Radiation.

Radical radiotherapy, often in combination with hormone ablation, is a safe and effective treatment option for localised or locally-advanced prostate cancer. However, up to 30% of patients with locally advanced PCa will go on to develop biochemical failure, within 5 years, following initial radiotherapy. Improving radiotherapy response is clinically important since patients exhibiting biochemical failure develop castrate-resistant metastatic disease for which there is no curative therapy and median survival is 8-18 months. The aim of this research was to determine if loss of PTEN (highly prevalent in advanced prostate cancer) is a novel therapeutic target in the treatment of advanced prostate cancer. Previous work has demonstrated PTEN-deficient cells are sensitised to inhibitors of ATM, a key regulator in the response to DSBs. Here, we have shown the role of PTEN in cellular response to IR was both complex and context-dependent. Secondly, we have confirmed ATM inhibition in PTEN-depleted cell models, enhances ionising radiation-induced cell killing with minimal toxicity to normal prostate RWPE-1 cells. Furthermore, combined treatment significantly inhibited PTEN-deficient tumour growth compared to PTEN-expressing counterparts, with minimal toxicity observed. We have further shown PTEN loss is accompanied by increased endogenous levels of ROS and DNA damage. Taken together, these findings provide pre-clinical data for future clinical evaluation of ATM inhibitors as a neoadjuvant/adjuvant in combination with radiation therapy in prostate cancer patients harbouring PTEN mutations.

ACE: A Workbench Using Evolutionary Genetic Algorithms for Analyzing Association in TCGA.

Modern methods of acquiring molecular data have improved rapidly in recent years, making it easier for researchers to collect large volumes of information. However, this has increased the challenge of recognizing interesting patterns within the data. Atlas Correlation Explorer (ACE) is a user-friendly workbench for seeking associations between attributes in The Cancer Genome Atlas (TCGA) database. It allows any combination of clinical and genomic data streams to be searched using an evolutionary algorithm approach. To showcase ACE, we assessed which RNA sequencing transcripts were associated with estrogen receptor (ESR1) in the TCGA breast cancer cohort. The analysis revealed already well-established associations with XBP1 and FOXA1, but also identified a strong association with CT62, a potential immunotherapeutic target with few previous associations with breast cancer. In conclusion, ACE can produce results for very large searches in a short time and will serve as an increasingly useful tool for biomarker discovery in the big data era. SIGNIFICANCE: ACE uses an evolutionary algorithm approach to perform large searches for associations between any combinations of data in the TCGA database.

Cytoplasmic FLIP(S) and nuclear FLIP(L) mediate resistance of castrate-resistant prostate cancer to apoptosis induced by IAP antagonists.

Expression of tumor necrosis factor-α (TNFα) in the serum of prostate cancer patients is associated with poorer outcome and progression to castrate-resistant (CRPC) disease. TNFα promotes the activity of NFκB, which regulates a number of anti-apoptotic and proinflammatory genes, including those encoding the inhibitor of apoptosis proteins (IAPs); however, in the presence of IAP antagonists, TNFα can induce cell death. In the presence of recombinant or macrophage-derived TNFα, we found that IAP antagonists triggered degradation of cIAP1 and induced formation of Complex-IIb, consisting of caspase-8, FADD and RIPK1 in CRPC models; however, no, or modest levels of apoptosis were induced. This resistance was found to be mediated by both the long (L) and short (S) splice forms of the caspase-8 inhibitor, FLIP, another NFκB-regulated protein frequently overexpressed in CRPC. By decreasing FLIP expression at the post-transcriptional level in PC3 and DU145 cells (but not VCaP), the Class-I histone deacetylase (HDAC) inhibitor Entinostat promoted IAP antagonist-induced cell death in these models in a manner dependent on RIPK1, FADD and Caspase-8. Of note, Entinostat primarily targeted the nuclear rather than cytoplasmic pool of FLIP(L). While the cytoplasmic pool of FLIP(L) was highly stable, the nuclear pool was more labile and regulated by the Class-I HDAC target Ku70, which we have previously shown regulates FLIP stability. The efficacy of IAP antagonist (TL32711) and Entinostat combination and their effects on cIAP1 and FLIP respectively were confirmed in vivo, highlighting the therapeutic potential for targeting IAPs and FLIP in proinflammatory CRPC.

Protocols for Studies on Genetically Engineered Mouse Models in Prostate Cancer.

Cancer studies have entered an era that is heavily focused on the contribution of the tumor microenvironment. For this reason, in vivo experimentation in an immunodeficient model system is no longer fit for purpose. As a consequence, numerous genetically engineered mouse models (GEMMs) which self-develop tumors have been developed to allow experiments to be performed in a fully immunocompetent setting. One of the most commonly used technologies is Cre-loxP recombination due to its unique ability to control target gene expression in a specified tissue type. However, the major limitation of these models remains the inability to generate sufficient numbers of age-matched mice for a synchronized experimental start date. For this reason, the derivation of cell lines from genetically modified murine prostate tissue is desirable and allows for the generation of syngeneic models via subcutaneous or orthotopic injection.

Molecular Subgroup of Primary Prostate Cancer Presenting with Metastatic Biology.

BACKGROUND: Approximately 4-25% of patients with early prostate cancer develop disease recurrence following radical prostatectomy. OBJECTIVE: To identify a molecular subgroup of prostate cancers with metastatic potential at presentation resulting in a high risk of recurrence following radical prostatectomy. DESIGN, SETTING, AND PARTICIPANTS: Unsupervised hierarchical clustering was performed using gene expression data from 70 primary resections, 31 metastatic lymph nodes, and 25 normal prostate samples. Independent assay validation was performed using 322 radical prostatectomy samples from four sites with a mean follow-up of 50.3 months. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Molecular subgroups were identified using unsupervised hierarchical clustering. A partial least squares approach was used to generate a gene expression assay. Relationships with outcome (time to biochemical and metastatic recurrence) were analysed using multivariable Cox regression and log-rank analysis. RESULTS AND LIMITATIONS: A molecular subgroup of primary prostate cancer with biology similar to metastatic disease was identified. A 70-transcript signature (metastatic assay) was developed and independently validated in the radical prostatectomy samples. Metastatic assay positive patients had increased risk of biochemical recurrence (multivariable hazard ratio [HR] 1.62 [1.13-2.33]; p=0.0092) and metastatic recurrence (multivariable HR=3.20 [1.76-5.80]; p=0.0001). A combined model with Cancer of the Prostate Risk Assessment post surgical (CAPRA-S) identified patients at an increased risk of biochemical and metastatic recurrence superior to either model alone (HR=2.67 [1.90-3.75]; p<0.0001 and HR=7.53 [4.13-13.73]; p<0.0001, respectively). The retrospective nature of the study is acknowledged as a potential limitation. CONCLUSIONS: The metastatic assay may identify a molecular subgroup of primary prostate cancers with metastatic potential. PATIENT SUMMARY: The metastatic assay may improve the ability to detect patients at risk of metastatic recurrence following radical prostatectomy. The impact of adjuvant therapies should be assessed in this higher-risk population.

Challenging the Cancer Molecular Stratification Dogma: Intratumoral Heterogeneity Undermines Consensus Molecular Subtypes and Potential Diagnostic Value in Colorectal Cancer.

PURPOSE: A number of independent gene expression profiling studies have identified transcriptional subtypes in colorectal cancer with potential diagnostic utility, culminating in publication of a colorectal cancer Consensus Molecular Subtype classification. The worst prognostic subtype has been defined by genes associated with stem-like biology. Recently, it has been shown that the majority of genes associated with this poor prognostic group are stromal derived. We investigated the potential for tumor misclassification into multiple diagnostic subgroups based on tumoral region sampled. EXPERIMENTAL DESIGN: We performed multiregion tissue RNA extraction/transcriptomic analysis using colorectal-specific arrays on invasive front, central tumor, and lymph node regions selected from tissue samples from 25 colorectal cancer patients. RESULTS: We identified a consensus 30-gene list, which represents the intratumoral heterogeneity within a cohort of primary colorectal cancer tumors. Using a series of online datasets, we showed that this gene list displays prognostic potential HR = 2.914 (confidence interval 0.9286-9.162) in stage II/III colorectal cancer patients, but in addition, we demonstrated that these genes are stromal derived, challenging the assumption that poor prognosis tumors with stem-like biology have undergone a widespread epithelial-mesenchymal transition. Most importantly, we showed that patients can be simultaneously classified into multiple diagnostically relevant subgroups based purely on the tumoral region analyzed. CONCLUSIONS: Gene expression profiles derived from the nonmalignant stromal region can influence assignment of colorectal cancer transcriptional subtypes, questioning the current molecular classification dogma and highlighting the need to consider pathology sampling region and degree of stromal infiltration when employing transcription-based classifiers to underpin clinical decision making in colorectal cancer. Clin Cancer Res; 22(16); 4095-104. ©2016 AACRSee related commentary by Morris and Kopetz, p. 3989.

PTEN deficiency promotes macrophage infiltration and hypersensitivity of prostate cancer to IAP antagonist/radiation combination therapy.

PTEN loss is prognostic for patient relapse post-radiotherapy in prostate cancer (CaP). Infiltration of tumor-associated macrophages (TAMs) is associated with reduced disease-free survival following radical prostatectomy. However, the association between PTEN loss, TAM infiltration and radiotherapy response of CaP cells remains to be evaluated. Immunohistochemical and molecular analysis of surgically-resected Gleason 7 tumors confirmed that PTEN loss correlated with increased CXCL8 expression and macrophage infiltration. However PTEN status had no discernable correlation with expression of other inflammatory markers by CaP cells, including TNF-α. In vitro, exposure to conditioned media harvested from irradiated PTEN null CaP cells induced chemotaxis of macrophage-like THP-1 cells, a response partially attenuated by CXCL8 inhibition. Co-culture with THP-1 cells resulted in a modest reduction in the radio-sensitivity of DU145 cells. Cytokine profiling revealed constitutive secretion of TNF-α from CaP cells irrespective of PTEN status and IR-induced TNF-α secretion from THP-1 cells. THP-1-derived TNF-α increased NFκB pro-survival activity and elevated expression of anti-apoptotic proteins including cellular inhibitor of apoptosis protein-1 (cIAP-1) in CaP cells, which could be attenuated by pre-treatment with a TNF-α neutralizing antibody. Treatment with a novel IAP antagonist, AT-IAP, decreased basal and TNF-α-induced cIAP-1 expression in CaP cells, switched TNF-α signaling from pro-survival to pro-apoptotic and increased radiation sensitivity of CaP cells in co-culture with THP-1 cells. We conclude that targeting cIAP-1 can overcome apoptosis resistance of CaP cells and is an ideal approach to exploit high TNF-α signals within the TAM-rich microenvironment of PTEN-deficient CaP cells to enhance response to radiotherapy.

CD44-mediated activation of α5ß1-integrin, cortactin and paxillin signaling underpins adhesion of basal-like breast cancer cells to endothelium and fibronectin-enriched matrices.

CD44 expression is elevated in basal-like breast cancer (BLBC) tissue, and correlates with increased efficiency of distant metastasis in patients and experimental models. We sought to characterize mechanisms underpinning CD44-promoted adhesion of BLBC cells to vascular endothelial monolayers and extracellular matrix (ECM) substrates. Stimulation with hyaluronan (HA), the native ligand for CD44, increased expression and activation of ß1-integrin receptors, and increased α5-integrin subunit expression. Adhesion assays confirmed that CD44-signalling potentiated BLBC cell adhesion to endothelium and Fibronectin in an α5B1-integrin-dependent mechanism. Co-immunoprecipitation experiments confirmed HA-promoted association of CD44 with talin and the ß1-integrin chain in BLBC cells. Knockdown of talin inhibited CD44 complexing with ß1-integrin and repressed HA-induced, CD44-mediated activation of ß1-integrin receptors. Immunoblotting confirmed that HA induced rapid phosphorylation of cortactin and paxillin, through a CD44-dependent and ß1-integrin-dependent mechanism. Knockdown of CD44, cortactin or paxillin independently attenuated the adhesion of BL-BCa cells to endothelial monolayers and Fibronectin. Accordingly, we conclude that CD44 induced, integrin-mediated signaling not only underpins efficient adhesion of BLBC cells to BMECs to facilitate extravasation but initiates their adhesion to Fibronectin, enabling penetrant cancer cells to adhere more efficiently to underlying Fibronectin-enriched matrix present within the metastatic niche.

The prognostic value of the stem-like group in colorectal cancer using a panel of immunohistochemistry markers.

Colorectal cancer (CRC) is the second leading cause of cancer-related deaths in the Western world. It is becoming increasingly clear that CRC is a diverse disease, as exemplified by the identification of subgroups of CRC tumours that are driven by distinct biology. Recently, a number of studies have begun to define panels of diagnostically relevant markers to align patients into individual subgroups in an attempt to give information on prognosis and treatment response. We examined the immunohistochemical expression profile of 18 markers, each representing a putative role in cancer development, in 493 primary colorectal carcinomas using tissue microarrays. Through unsupervised clustering in stage II cancers, we identified two cluster groups that are broadly defined by inflammatory or immune-related factors (CD3, CD8, COX-2 and FOXP3) and stem-like factors (CD44, LGR5, SOX2, OCT4). The expression of the stem-like group markers was associated with a significantly worse prognosis compared to cases with lower expression. In addition, patients classified in the stem-like subgroup displayed a trend towards a benefit from adjuvant treatment. The biologically relevant and poor prognostic stem-like group could also be identified in early stage I cancers, suggesting a potential opportunity for the identification of aggressive tumors at a very early stage of the disease.

CD44 increases the efficiency of distant metastasis of breast cancer.

Metastasis is the predominant cause of death from cancer yet we have few biomarkers to predict patients at increased risk of metastasis and are unable to effectively treat disseminated disease. Analysis of 448 primary breast tumors determined that expression of the hylauronan receptor CD44 associated with high grade (p = 0.046), ER- (p = 0.001) and PR-negative tumors (p = 0.029), and correlated with increased distant recurrence and reduced disease-free survival in patients with lymph-node positive or large tumors. To determine its functional role in distant metastasis, CD44 was knocked-down in MDA-MB-231 cells using two independent shRNA sequences. Loss of CD44 attenuated tumor cell adhesion to endothelial cells and reduced cell invasion but did not affect proliferation in vitro. To verify the importance of CD44 to post-intravasation events, tumor formation was assessed by quantitative in vivo imaging and post-mortem tissue analysis following an intra-cardiac injection of transfected cells. CD44 knock-down increased survival and decreased overall tumor burden at multiple sites, including the skeleton in vivo. We conclude that elevated CD44 expression on tumour cells within the systemic circulation increases the efficiency of post-intravasation events and distant metastasis in vivo, consistent with its association with increased distant recurrence and reduced disease-free survival in patients.

Tumor-derived CXCL8 signaling augments stroma-derived CCL2-promoted proliferation and CXCL12-mediated invasion of PTEN-deficient prostate cancer cells.

Impaired PTEN function is a genetic hallmark of aggressive prostate cancers (CaP) and is associated with increased CXCL8 expression and signaling. The current aim was to further characterize biological responses and mechanisms underpinning CXCL8-promoted progression of PTEN-depleted prostate cancer, focusing on characterizing the potential interplay between CXCL8 and other disease-promoting chemokines resident within the prostate tumor microenvironment. Autocrine CXCL8-stimulation (i) increased expression of CXCR1 and CXCR2 in PTEN-deficient CaP cells suggesting a self-potentiating signaling axis and (ii) induced expression of CXCR4 and CCR2 in PTEN-wild-type and PTEN-depleted CaP cells. In contrast, paracrine CXCL8 signaling induced expression and secretion of the chemokines CCL2 and CXCL12 from prostate stromal WPMY-1 fibroblasts and monocytic macrophage-like THP-1 cells. In vitro studies demonstrated functional co-operation of tumor-derived CXCL8 with stromal-derived chemokines. CXCL12-induced migration of PC3 cells and CCL2-induced proliferation of prostate cancer cells were dependent upon intrinsic CXCL8 signaling within the prostate cancer cells. For example, in co-culture experiments, CXCL12/CXCR4 signaling but not CCL2/CCR2 signaling supported fibroblast-mediated migration of PC3 cells while CXCL12/CXCR4 and CCL2/CCR2 signaling underpinned monocyte-enhanced migration of PC3 cells. Combined inhibition of both CXCL8 and CXCL12 signaling was more effective in inhibiting fibroblast-promoted cell motility while repression of CXCL8 attenuated CCL2-promoted proliferation of prostate cancer cells. We conclude that tumor-derived CXCL8 signaling from PTEN-deficient tumor cells increases the sensitivity and responsiveness of CaP cells to stromal chemokines by concurrently upregulating receptor expression in cancer cells and inducing stromal chemokine synthesis. Combined chemokine targeting may be required to inhibit their multi-faceted actions in promoting the invasion and proliferation of aggressive CaP.

TBX2 represses CST6 resulting in uncontrolled legumain activity to sustain breast cancer proliferation: a novel cancer-selective target pathway with therapeutic opportunities.

TBX2 is an oncogenic transcription factor known to drive breast cancer proliferation. We have identified the cysteine protease inhibitor Cystatin 6 (CST6) as a consistently repressed TBX2 target gene, co-repressed through a mechanism involving Early Growth Response 1 (EGR1). Exogenous expression of CST6 in TBX2-expressing breast cancer cells resulted in significant apoptosis whilst non-tumorigenic breast cells remained unaffected. CST6 is an important tumor suppressor in multiple tissues, acting as a dual protease inhibitor of both papain-like cathepsins and asparaginyl endopeptidases (AEPs) such as Legumain (LGMN). Mutation of the CST6 LGMN-inhibitory domain completely abrogated its ability to induce apoptosis in TBX2-expressing breast cancer cells, whilst mutation of the cathepsin-inhibitory domain or treatment with a pan-cathepsin inhibitor had no effect, suggesting that LGMN is the key oncogenic driver enzyme. LGMN activity assays confirmed the observed growth inhibitory effects were consistent with CST6 inhibition of LGMN. Knockdown of LGMN and the only other known AEP enzyme (GPI8) by siRNA confirmed that LGMN was the enzyme responsible for maintaining breast cancer proliferation. CST6 did not require secretion or glycosylation to elicit its cell killing effects, suggesting an intracellular mode of action. Finally, we show that TBX2 and CST6 displayed reciprocal expression in a cohort of primary breast cancers with increased TBX2 expression associating with increased metastases. We have also noted that tumors with altered TBX2/CST6 expression show poor overall survival. This novel TBX2-CST6-LGMN signaling pathway, therefore, represents an exciting opportunity for the development of novel therapies to target TBX2 driven breast cancers.